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The livestock sector, essential for maintaining food supply and security, encounters numerous obstacles as a result of climate change. Rising global populations exacerbate competition for natural resources, affecting feed quality and availability, heightening livestock disease risks, increasing heat stress, and contributing to biodiversity loss. Although various management and dietary interventions exist to alleviate these impacts, they often offer only short-lived solutions. We must take a more comprehensive approach to understanding how animals adapt to and endure their environments. One such approach is quantifying transcriptomes under different environments, which can uncover underlying pathways essential for livestock adaptation. This review explores the progress and techniques in studies that apply gene expression analysis to livestock production systems, focusing on their adaptation to climate change. We also attempt to identify various biomarkers and transcriptomic differences between species and pure/crossbred animals. Looking ahead, integrating emerging technologies such as spatialomics could further accelerate genetic improvements, enabling more thermoresilient and productive livestock in response to future climate fluctuations. Ultimately, insights from these studies will help optimize livestock production systems by identifying thermoresilient/desired animals for use in precise breeding programs to counter climate change.

Heat stress impacts ruminant livestock production on varied levels in this alarming climate breakdown scenario. The drastic effects of the global climate change-associated heat stress in ruminant livestock demands constructive evaluation of animal performance bordering on effective monitoring systems. In this climate-smart digital age, adoption of advanced and developing Artificial Intelligence (AI) technologies is gaining traction for efficient heat stress management. AI has widely penetrated the climate sensitive ruminant livestock sector due to its promising and plausible scope in assessing production risks and the climate resilience of ruminant livestock. Significant improvement has been achieved alongside the adoption of novel AI algorithms to evaluate the performance of ruminant livestock. These AI-powered tools have the robustness and competence to expand the evaluation of animal performance and help in minimising the production losses associated with heat stress in ruminant livestock. Advanced heat stress management through automated monitoring of heat stress in ruminant livestock based on behaviour, physiology and animal health responses have been widely accepted due to the evolution of technologies like machine learning (ML), neural networks and deep learning (DL). The AI-enabled tools involving automated data collection, pre-processing, data wrangling, development of appropriate algorithms, and deployment of models assist the livestock producers in decision-making based on real-time monitoring and act as early-stage warning systems to forecast disease dynamics based on prediction models. Due to the convincing performance, precision, and accuracy of AI models, the climate-smart livestock production imbibes AI technologies for scaled use in the successful reducing of heat stress in ruminant livestock, thereby ensuring sustainable livestock production and safeguarding the global economy.

A comprehensive study was conducted to assess the effects of seasonal transition and temperature humidity index (THI) on the adaptive responses in crossbred dairy cows reared in a tropical savanna region. A total of 40 lactating dairy cattle reared by small-scale dairy farmers in Bengaluru, India, were selected for this study. The research period comprised the transitioning season of summer to monsoon, wherein all traits were recorded at two points, one representing late summer (June) and the other early monsoon (July). A set of extensive variables representing physiological responses (pulse rate, respiration rate, rectal temperature, skin surface temperature), hematological responses (hematological profile), production (test day milk yield, milk composition) and molecular patterns (PBMC mRNA relative expression of selective stress response genes) were assessed. A significant effect of seasonal transition was identified on respiration rate (RR), skin surface temperature, mean platelet volume (MPV), platelet distribution width (PDWc), test day milk yield and on milk composition variables (milk density, lactose, solids-not-fat (SNF) and salts). The THI had a significant effect on RR, skin surface temperature, platelet count (PLT), plateletcrit (PCT) and PDWc. Lastly, THI and/or seasonal transition significantly affected the relative PBMC mRNA expression of heat shock protein 70 (HSP70), interferon beta (IFNβ), IFNγ, tumor necrosis factor alpha (TNFα), growth hormone (GH) and insulin-like growth factor-1 (IGF-1) genes. The results from this study reveal environmental sensitivity of novel physiological traits and gene expressions to climatic stressors, highlighting their potential as THI-independent heat stress biomarkers.

Non-invasive methods of detecting heat stress magnitude for livestock is gaining momentum in the context of global climate change. Therefore, the objective of this review is to focus on the synthesis information pertaining to recent efforts to develop heat stress detection systems for livestock based on multiple behavioral and physiological responses. There are a number of approaches to quantify farm animal heat stress response, and from an animal welfare point of view, these can be categorized as invasive and non-invasive approaches. The concept of a non-invasive approach to assess heat stress primarily looks into behavioral and physiological responses which can be monitored without any human interference or additional stress on the animal. Bioclimatic thermal indices can be considered as the least invasive approach to assess and/or predict the level of heat stress in livestock. The quantification and identification of the fecal microbiome in heat-stressed farm animals is one of the emerging techniques which could be effectively correlated with animal adaptive responses. Further, tremendous progress has been made in the last decade to quantify the classical heat stress endocrine marker, cortisol, non-invasively in the feces, urine, hair, saliva and milk of farm animals. In addition, advanced technologies applied for the real-time analysis of cardinal signs such as sounds through microphones, behavioral images, videos through cameras, and data stalking body weight and measurements might provide deeper insights towards improving biological metrics in livestock exposed to heat stress. Infrared thermography (IRT) can be considered another non-invasive modern tool to assess the stress response, production, health, and welfare status in farm animals. Various remote sensing technologies such as ear canal sensors, rumen boluses, rectal and vaginal probes, IRT, and implantable microchips can be employed in grazing animals to assess the quantum of heat stress. Behavioral responses and activity alterations to heat stress in farm animals can be monitored using accelerometers, Bluetooth technology, global positioning systems (GPSs) and global navigation satellite systems (GNSSs). Finally, machine learning offers a scalable solution in determining the heat stress response in farm animals by utilizing data from different sources such as hardware sensors, e.g., pressure sensors, thermistors, IRT sensors, facial recognition machine vision sensors, radio frequency identification, accelerometers, and microphones. Thus, the recent advancements in recording behavior and physiological responses offer new scope to quantify farm animals’ heat stress response non-invasively. These approaches could have greater applications in not only determining climate resilience in farm animals but also providing valuable information for defining suitable and accurate amelioration strategies to sustain their production.

The TATA-box binding protein associated factor 1 (TAF1) is part of the TFIID complex that plays a key role during the initiation of transcription. Variants of TAF1 are associated with neurodevelopmental disorders. Previously, we found that CRISPR/Cas9 based editing of the TAF1 gene disrupts the morphology of the cerebral cortex and blunts the expression as well as the function of the CaV3.1 (T-type) voltage gated calcium channel. Here, we tested the efficacy of SAK3 (ethyl 8′-methyl-2′, 4-dioxo-2-(piperidin-1-yl)-2′H-spiro [cyclopentane-1, 3′-imidazo [1, 2-a] pyridine]-2-ene-3-carboxylate), a T-type calcium channel enhancer, in an animal model of TAF1 intellectual disability (ID) syndrome. At post-natal day 3, rat pups were subjected to intracerebroventricular (ICV) injection of either gRNA-control or gRNA-TAF1 CRISPR/Cas9 viruses. At post-natal day 21, the rat pups were given SAK3 (0.25 mg/kg, p.o.) or vehicle for 14 days (i.e. till post-natal day 35) and then subjected to behavioral, morphological, and molecular studies. Oral administration of SAK3 (0.25 mg/kg, p.o.) significantly rescued locomotion abnormalities associated with TAF1 gene editing. SAK3 treatment prevented the loss of cortical neurons and GFAP-positive astrocytes observed after TAF1 gene editing. In addition, SAK3 protected cells from apoptosis. SAK3 also restored the Brain-derived neurotrophic factor/protein kinase B/Glycogen Synthase Kinase 3 Beta (BDNF/AKT/GSK3β) signaling axis in TAF1 edited animals. Finally, SAK3 normalized the levels of three GSK3β substrates - CaV3.1, FOXP2, and CRMP2. We conclude that the T-type calcium channel enhancer SAK3 is beneficial against the deleterious effects of TAF1 gene-editing, in part, by stimulating the BDNF/AKT/GSK3β signaling pathway.

T-type calcium channels, in the central nervous system, are involved in the pathogenesis of many neurodegenerative diseases, including TAF1 intellectual disability syndrome (TAF1 ID syndrome). Here, we evaluated the efficacy of a novel T-type Ca2+ channel enhancer, SAK3 (ethyl 8′-methyl-2′, 4-dioxo-2-(piperidin-1-yl)-2′H-spiro [cyclopentane-1, 3′-imidazo [1, 2-a] pyridine]-2-ene-3-carboxylate) in an animal model of TAF1 ID syndrome. At post-natal day 3, rat pups were subjected to intracerebroventricular (ICV) injection of either gRNA-control or gRNA-TAF1 CRISPR/Cas9 viruses. At post-natal day 21 animals were given SAK3 (0.25 mg/kg, p.o.) or vehicle up to post-natal day 35 (i.e. 14 days). Rats were subjected to behavioral, morphological, electrophysiological, and molecular studies. Oral administration of SAK3 (0.25 mg/kg, p.o.) significantly rescued the behavior abnormalities in beam walking test and open field test caused by TAF1 gene editing. We observed an increase in calbindin-positive Purkinje cells and GFAP-positive astrocytes as well as a decrease in IBA1-positive microglia cells in SAK3-treated animals. In addition, SAK3 protected the Purkinje and granule cells from apoptosis induced by TAF-1 gene editing. SAK3 also restored the excitatory post synaptic current (sEPSCs) in TAF1 edited Purkinje cells. Finally, SAK3 normalized the BDNF/AKT signaling axis in TAF1 edited animals. Altogether, these observations suggest that SAK3 could be a novel therapeutic agent for TAF1 ID syndrome.

Abstract Background Mitochondrial dysfunction and oxidative stress are the main toxic events leading to dopaminergic neuronal death in Parkinson’s disease (PD) and identified as vital objective for therapeutic intercession. This study investigated the neuro-protective effects of the demethoxycurcumin (DMC), a derivative of curcumin against rotenone induced neurotoxicity. Methods SH-SY5Y neuroblastoma cells are divided into four experimental groups: untreated cells, cells incubated with rotenone (100 nM), cells treated with DMC (50 nM) + rotenone (100 nM) and DMC alone treated. 24 h after treatment with rotenone and 28 h after treatment with DMC, cell viability was assessed using the MTT assay, and levels of ROS and MMP, plus expression of apoptotic protein were analysed. Results Rotenone induced cell death in SH-SY5Y cells was significantly reduced by DMC pretreatment in a dose-dependent manner, indicating the potent neuroprotective effects of DMC. Rotenone treatment significantly increases the levels of ROS, loss of MMP, release of Cyt-c and expression of pro-apoptotic markers and decreases the expression of anti-apoptotic markers. Conclusions Even though the results of the present study indicated that the DMC may serve as a potent therapeutic agent particularly for the treatment of neurodegenerative diseases like PD, further pre-clinical and clinical studies are required.

This review attempted to collate and synthesize information on goat welfare and production constraints during heat stress exposure. Among the farm animals, goats arguably are considered the best-suited animals to survive in tropical climates. Heat stress was found to negatively influence growth, milk and meat production and compromised the immune response, thereby significantly reducing goats’ welfare under extensive conditions and transportation. Although considered extremely adapted to tropical climates, their production can be compromised to cope with heat stress. Therefore, information on goat adaptation and production performance during heat exposure could help assess their welfare. Such information would be valuable as the farming communities are often struggling in their efforts to assess animal welfare, especially in tropical regions. Broadly three aspects must be considered to ensure appropriate welfare in goats, and these include (i) housing and environment; (ii) breeding and genetics and (iii) handling and transport. Apart from these, there are a few other negative welfare factors in goat rearing, which differ across the production system being followed. Such negative practices are predominant in extensive systems and include nutritional stress, limited supply of good quality water, climatic extremes, parasitic infestation and lameness, culminating in low production, reproduction and high mortality rates. Broadly two types of methodologies are available to assess welfare in goats in these systems: (i) animal-based measures include behavioral measurements, health and production records and disease symptoms; (ii) resources based and management-based measures include stocking density, manpower, housing conditions and health plans. Goat welfare could be assessed based on several indicators covering behavioral, physical, physiological and productive responses. The important indicators of goat welfare include agonistic behavior, vocalization, skin temperature, body condition score (BCS), hair coat conditions, rectal temperature, respiration rate, heart rate, sweating, reduced growth, reduced milk production and reduced reproductive efficiency. There are also different approaches available by which the welfare of goats could be assessed, such as naturalistic, functional and subjective approaches. Thus, assessing welfare in goats at every production stage is a prerequisite for ensuring appropriate production in this all-important species to guarantee optimum returns to the marginal and subsistence farmers.

Following transduction with a retrovirus (SF1MIH) expressing both the multidrug resistance 1 (MDR1) and O6-alkylguanine-DNA-alkyltransferase (ATase) proteins, human erythroleukaemic progenitor (K562) cells were isolated which were resistant to killing by the MDR1 substrate, colchicine. In colony-forming survival assays, K562-SF1MIH cells exhibited resistance to colchicine and doxorubicin, as well as to the O6-alkylating agents N-Methyl-N-nitrosourea (MNU) and temozolomide. Furthermore, the resistance to doxorubicin was abolished by preincubation with the MDR1 inhibitor verapamil while resistance to MNU was ablated by the specific ATase inactivator, O6-benzylguanine (O6-beG) confirming that resistance to doxorubicin and MNU was conferred by MDR1 and ATase, respectively. When K562-SF1MIH were exposed to combinations of colchicine and MNU or doxorubicin and temozolomide, simultaneous resistance to these agents was observed. Thus, transduction of K562 with SF1MIH conferred dual resistance to these cells. These data offer the prospect of designing vectors that will confer resistance to entire regimens of chemotherapy rather than just to individual components of such drug cocktails, thereby substantially increasing the efficacy of therapy. Furthermore, the use of such dual expression constructs is likely to be highly informative for the design of effective in vivo selection protocols, an issue likely to make a major impact in a clinical context in gene therapy in the near future. [ABSTRACT FROM AUTHOR]

Abstract Background A number of gene therapy applications would benefit from vectors capable of expressing multiple genes. In this study we explored the feasibility and efficiency of expressing two or three transgenes in HIV-1 based lentiviral vector. Bicistronic and tricistronic self-inactivating lentiviral vectors were constructed employing the internal ribosomal entry site (IRES) sequence of encephalomyocarditis virus (EMCV) and/or foot-and-mouth disease virus (FMDV) cleavage factor 2A. We employed enhanced green fluorescent protein (eGFP), O6-methylguanine-DNA-methyltransferase (MGMT), and homeobox transcription factor HOXB4 as model genes and their expression was detected by appropriate methods including fluorescence microscopy, flow cytometry, immunocytochemistry, biochemical assay, and western blotting. Results All the multigene vectors produced high titer virus and were able to simultaneously express two or three transgenes in transduced cells. However, the level of expression of individual transgenes varied depending on: the transgene itself; its position within the construct; the total number of transgenes expressed; the strategy used for multigene expression and the average copy number of pro-viral insertions. Notably, at limiting MOI, the expression of eGFP in a bicistronic vector based on 2A was ~4 times greater than that of an IRES based vector. Conclusion The small and efficient 2A sequence can be used alone or in combination with an IRES for the construction of multicistronic lentiviral vectors which can express encoded transgenes at functionally relevant levels in cells containing an average of one pro-viral insert.

Introduction: Malaria is a vector-borne disease that initially manifests as fever, headache, and chills. The illness could progress to more severe conditions, including lethargy, impaired consciousness, convulsions, shortness of breath, blood in urine, jaundice, and haemorrhage if left untreated. The risk of contracting malaria is considerably heightened in specific occupational settings, particularly among forest rangers, following frequent exposure to natural habitats. Consequently, advancing the understanding of malaria and emphasising how specific occupational environments (including those of forest rangers) contribute to disease risk and management is imperative., Objective: The present study aims to determine the factors associated with malaria infection among forest rangers by systematically reviewing electronic articles from three databases (EBSCOhost, ScienceDirect, and ResearchGate)., Methods: The current review was prepared based on the updated preferred reporting items for systematic reviews and meta-analyses (PRISMA) guidelines. First, three independent reviewers screened the titles and abstracts of the data collected. The information was then stored in Endnote20 based on the inclusion and exclusion criteria. The articles were critically appraised with the mixed methods appraisal tool (MMAT) to assess their quality., Result: A total of 103, 31, and 51 articles from EBSCOhost, ScienceDirect, and ResearchGate, respectively, were selected, resulting in 185 unique hits. Nevertheless, only 63 full-text publications were assessed following a rigorous selection screening, from which only five were included in the final review. The studies revealed that several factors contribute to malaria infection among forest rangers. The parameters were classified into sociodemographic, individual, and living condition-related., Conclusion: A better understanding of malaria progresses and identifying its potential risk factors is essential to impact worker well-being. The findings might be utilised to improve malaria infection prevention programme implementations, hence maximising their success. Pre-employment and regular health screenings could also aid in evaluating and identifying potential risks for malaria infection among forest rangers., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Dapari et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Metabolic disruption commonly follows Anterior Cruciate Ligament Reconstruction (ACLR) surgery. Brief exposure to low amplitude and frequency pulsed electromagnetic fields (PEMFs) has been shown to promote in vitro and in vivo murine myogeneses via the activation of a calcium-mitochondrial axis conferring systemic metabolic adaptations. This randomized-controlled pilot trial sought to detect local changes in muscle structure and function using MRI, and systemic changes in metabolism using plasma biomarker analyses resulting from ACLR, with or without accompanying PEMF therapy., Methods: 20 patients requiring ACLR were randomized into two groups either undergoing PEMF or sham exposure for 16 weeks following surgery. The operated thighs of 10 patients were exposed weekly to PEMFs (1 ​mT for 10 ​min) for 4 months following surgery. Another 10 patients were subjected to sham exposure and served as controls to allow assessment of the metabolic repercussions of ACLR and PEMF therapy. Blood samples were collected prior to surgery and at 16 weeks for plasma analyses. Magnetic resonance data were acquired at 1 and 16 weeks post-surgery using a Siemens 3T Tim Trio system. Phosphorus ( 31 P) Magnetic Resonance Spectroscopy (MRS) was utilized to monitor changes in high-energy phosphate metabolism (inorganic phosphate (P i ), adenosine triphosphate (ATP) and phosphocreatine (PCr)) as well as markers of membrane synthesis and breakdown (phosphomonoesters (PME) and phosphodiester (PDE)). Quantitative Magnetization Transfer (qMT) imaging was used to elucidate changes in the underlying tissue structure, with T1-weighted and 2-point Dixon imaging used to calculate muscle volumes and muscle fat content., Results: Improvements in markers of high-energy phosphate metabolism including reductions in ΔP i /ATP, P i /PCr and (P i  ​+ ​PCr)/ATP, and membrane kinetics, including reductions in PDE/ATP were detected in the PEMF-treated cohort relative to the control cohort at study termination. These were associated with reductions in the plasma levels of certain ceramides and lysophosphatidylcholine species. The plasma levels of biomarkers predictive of muscle regeneration and degeneration, including osteopontin and TNNT1, respectively, were improved, whilst changes in follistatin failed to achieve statistical significance. Liquid chromatography with tandem mass spectrometry revealed reductions in small molecule biomarkers of metabolic disruption, including cysteine, homocysteine, and methionine in the PEMF-treated cohort relative to the control cohort at study termination. Differences in measurements of force, muscle and fat volumes did not achieve statistical significance between the cohorts after 16 weeks post-ACLR., Conclusion: The detected changes suggest improvements in systemic metabolism in the post-surgical PEMF-treated cohort that accords with previous preclinical murine studies. PEMF-based therapies may potentially serve as a manner to ameliorate post-surgery metabolic disruptions and warrant future examination in more adequately powered clinical trials., The Translational Potential of This Article: Some degree of physical immobilisation must inevitably follow orthopaedic surgical intervention. The clinical paradox of such a scenario is that the regenerative potential of the muscle mitochondrial pool is silenced. The unmet need was hence a manner to maintain mitochondrial activation when movement is restricted and without producing potentially damaging mechanical stress. PEMF-based therapies may satisfy the requirement of non-invasively activating the requisite mitochondrial respiration when mobility is restricted for improved metabolic and regenerative recovery., Competing Interests: AFO, JF and CB are inventors on patent WO 2019/17863 A1, System, and Method for Applying Pulsed Electromagnetic Fields, AFO and JF are contributors to QuantumTx Pte. Ltd., which elaborates electromagnetic field devices for human use. JZL is currently an employee of QuantumTx Pte. Ltd, but at the time of writing the manuscript was an employee of the NUS, Department of Surgery. All other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)

Purpose: To compare the clinical outcomes of the routine use of 5-strand hamstring grafts (where possible) with those of 4-strand grafts in primary anterior cruciate ligament (ACL) reconstruction., Methods: A total of 64 patients were enrolled in a prospective randomized controlled study comparing the use of 5-strand and 4-strand semitendinosus-gracilis autografts in single bundle ACL reconstruction (n = 32 in each group). Four participants in each group were lost to follow-up and were excluded from the outcome analysis. The outcomes of 28 patients in the 5-strand group and 28 patients in the 4-strand group were analyzed. The diameters of all grafts were measured intraoperatively. Patients were assessed postoperatively at 2 years with objective assessments (anterior knee laxity using the KT-2000 arthrometer, Lachman test, pivot-shift test, hop test) and patient-reported outcome scores (Lysholm knee score, Knee Injury and Osteoarthritis Outcome Score, International Knee Documentation Committee subjective knee score, SF-36 physical and mental components, Tegner activity scale). Postoperative graft ruptures were also noted., Results: There were improvements in all outcome measures postoperatively regardless of the number of graft strands. When we compared the study and control groups, there were no significant differences in all subjective and objective outcome measures except the Knee Injury and Osteoarthritis Outcome Score symptoms score (5-strand group 93.3 ± 9.2 vs 4-strand group 86.2 ± 14.7, P = .04). The KT-2000 side-to-side difference was 2.79 ± 2.11 mm in the 5-strand group and 2.54 ± 1.75 mm in the 4-strand group (P = .63). The 5-strand study group had 2 graft ruptures at 1 year, whereas the 4-strand control group had one partial graft rupture at 6 months., Conclusions: At 2-year follow-up, the routine use of the 5-strand hamstring tendon autograft was not superior to that of the quadrupled or 4-strand graft in primary ACL reconstruction., Level of Evidence: Level I, prospective randomized controlled trial., (Copyright © 2020 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.)

TAF1/MRSX33 intellectual disability syndrome is an X-linked disorder caused by loss-of-function mutations in the TAF1 gene. How these mutations cause dysmorphology, hypotonia, intellectual and motor defects is unknown. Mouse models which have embryonically targeted TAF1 have failed, possibly due to TAF1 being essential for viability, preferentially expressed in early brain development, and intolerant of mutation. Novel animal models are valuable tools for understanding neuronal pathology. Here, we report the development and characterization of a novel animal model for TAF1 ID syndrome in which the TAF1 gene is deleted in embryonic rats using clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9) technology and somatic brain transgenesis mediated by lentiviral transduction. Rat pups, post-natal day 3, were subjected to intracerebroventricular (ICV) injection of either gRNA-control or gRNA-TAF1 vectors. Rats were subjected to a battery of behavioral tests followed by histopathological analyses of brains at post-natal day 14 and day 35. TAF1-edited rats exhibited behavioral deficits at both the neonatal and juvenile stages of development. Deletion of TAF1 lead to a hypoplasia and loss of the Purkinje cells. We also observed a decreased in GFAP positive astrocytes and an increase in Iba1 positive microglia within the granular layer of the cerebellum in TAF1-edited animals. Immunostaining revealed a reduction in the expression of the CaV3.1 T-type calcium channel. Abnormal motor symptoms in TAF1-edited rats were associated with irregular cerebellar output caused by changes in the intrinsic activity of the Purkinje cells due to loss of pre-synaptic CaV3.1. This animal model provides a powerful new tool for studies of neuronal dysfunction in conditions associated with TAF1 abnormalities and should prove useful for developing therapeutic strategies to treat TAF1 ID syndrome., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

A growing body of evidence suggests that inflammatory cytokines have a dualistic role in immunity. In this study, we sought to determine the direct effects of interferon-γ (IFN-γ) on the differentiation and maturation of human peripheral blood monocyte-derived dendritic cells (moDC). Here, we report that following differentiation of monocytes into moDC with granulocyte-macrophage colony-stimulating factor and interleukin-4, IFN-γ induces moDC maturation and up-regulates the co-stimulatory markers CD80/CD86/CD95 and MHC Class I, enabling moDC to effectively generate antigen-specific CD4(+) and CD8(+) T-cell responses for multiple viral and tumour antigens. Early exposure of monocytes to high concentrations of IFN-γ during differentiation promotes the formation of macrophages. However, under low concentrations of IFN-γ, monocytes continue to differentiate into dendritic cells possessing a unique gene-expression profile, resulting in impairments in subsequent maturation by IFN-γ or lipopolysaccharide and an inability to generate effective antigen-specific CD4(+) and CD8(+) T-cell responses. These findings demonstrate that IFN-γ imparts differential programmes on moDC that shape the antigen-specific T-cell responses they induce. Timing and intensity of exposure to IFN-γ can therefore determine the functional capacity of moDC., (Published 2014. This article is a U.S. Government work and is in the public domain in the USA.)

Fibroblast activation protein (FAP) is a candidate universal target antigen because it has been reported to be selectively expressed in nearly all solid tumors by a subset of immunosuppressive tumor stromal fibroblasts. We verified that 18/18 human tumors of various histologies contained pronounced stromal elements staining strongly for FAP, and hypothesized that targeting tumor stroma with FAP-reactive T cells would inhibit tumor growth in cancer-bearing hosts. T cells genetically engineered with FAP-reactive chimeric antigen receptors (CARs) specifically degranulated and produced effector cytokines upon stimulation with FAP or FAP-expressing cell lines. However, adoptive transfer of FAP-reactive T cells into mice bearing a variety of subcutaneous tumors mediated limited antitumor effects and induced significant cachexia and lethal bone toxicities in two mouse strains. We found that FAP was robustly expressed on PDGFR-α(+), Sca-1(+) multipotent bone marrow stromal cells (BMSCs) in mice, as well as on well-characterized, clinical-grade multipotent human BMSCs. Accordingly, both mouse and human multipotent BMSCs were recognized by FAP-reactive T cells. The lethal bone toxicity and cachexia observed after cell-based immunotherapy targeting FAP cautions against its use as a universal target. Moreover, the expression of FAP by multipotent BMSCs may point toward the cellular origins of tumor stromal fibroblasts.

Most systemic cancer therapies target tumor cells directly, although there is increasing interest in targeting the tumor stroma that can comprise a substantial portion of the tumor mass. We report here a synergy between two T-cell therapies, one directed against the stromal tumor vasculature and the other directed against antigens expressed on the tumor cell. Simultaneous transfer of genetically engineered syngeneic T cells expressing a chimeric antigen receptor targeting the VEGF receptor-2 (VEGFR2; KDR) that is overexpressed on tumor vasculature and T-cells specific for the tumor antigens gp100 (PMEL), TRP-1 (TYRP1), or TRP-2 (DCT) synergistically eradicated established B16 melanoma tumors in mice and dramatically increased the tumor-free survival of mice compared with treatment with either cell type alone or T cells coexpressing these two targeting molecules. Host lymphodepletion before cell transfer was required to mediate the antitumor effect. The synergistic antitumor response was accompanied by a significant increase in the infiltration and expansion and/or persistence of the adoptively transferred tumor antigen-specific T cells in the tumor microenvironment and thus enhanced their antitumor potency. The data presented here emphasize the possible beneficial effects of combining antiangiogenic with tumor-specific immunotherapeutic approaches for the treatment of patients with cancer., (©2013 AACR.)

Purpose: We investigated the feasibility of delivering the proinflammatory cytokine interleukin (IL)-12 into tumor using T cells genetically engineered to express a chimeric antigen receptor (CAR) against the VEGF receptor-2 (VEGFR-2)., Experimental Design: Two different strains of mice bearing five different established subcutaneous tumors were treated with syngeneic T cells cotransduced with an anti-VEGFR-2 CAR and a constitutively expressed single-chain murine IL-12 or an inducible IL-12 gene after host lymphodepletion. Tumor regression, survival of mice, and persistence of the transferred cells were evaluated., Results: Adoptive transfer of syngeneic T cells cotransduced with an anti-VEGFR-2 CAR and a constitutively expressing single-chain IL-12 resulted in the regression of five different established tumors of different histologies without the need for IL-2 administration. T cells transduced with either anti-VEGFR-2 CAR or single-chain IL-12 alone did not alter the tumor growth indicating that both of them had to be expressed in the same cell to mediate tumor regression. Anti-VEGFR-2 CAR and IL-12-cotransduced T cells infiltrated the tumors, expanded, and persisted for prolonged periods. The antitumor effect did not require the presence of host T and B cells but was dependent on host IL-12R-expressing cells. The anti-VEGFR-2 CAR changed the immunosuppressive tumor environment by altering/reducing both the systemic and the intratumoral CD11b(+)Gr1(+) myeloid suppressor cell subsets that expressed VEGFR-2., Conclusions: These results suggest that targeted delivery of IL-12 into the tumor environment with T cells redirected against VEGFR-2 is a promising approach for treating patients with a variety of solid tumor types.

Solid tumors are complex masses with a local microenvironment, or stroma, that supports tumor growth and progression. Among the diverse tumor-supporting stromal cells is a heterogeneous population of myeloid-derived cells. These cells are alternatively activated and contribute to the immunosuppressive environment of the tumor; overcoming their immunosuppressive effects may improve the efficacy of cancer immunotherapies. We recently found that engineering tumor-specific CD8(+) T cells to secrete the inflammatory cytokine IL-12 improved their therapeutic efficacy in the B16 mouse model of established melanoma. Here, we report the mechanism underlying this finding. Surprisingly, direct binding of IL-12 to receptors on lymphocytes or NK cells was not required. Instead, IL-12 sensitized bone marrow-derived tumor stromal cells, including CD11b(+)F4/80(hi) macrophages, CD11b(+)MHCII(hi)CD11c(hi) dendritic cells, and CD11b(+)Gr-1(hi) myeloid-derived suppressor cells, causing them to enhance the effects of adoptively transferred CD8(+) T cells. This reprogramming of myeloid-derived cells occurred partly through IFN-γ. Surprisingly, direct presentation of antigen to the transferred CD8(+) T cells by tumor was not necessary; however, MHCI expression on host cells was essential for IL-12-mediated antitumor enhancements. These results are consistent with a model in which IL-12 enhances the ability of CD8(+) T cells to collapse large vascularized tumors by triggering programmatic changes in otherwise suppressive antigen-presenting cells within tumors and support the use of IL-12 as part of immunotherapy for the treatment of solid tumors.

T-cell receptor (TCR) gene therapy enables for the rapid creation of antigen-specific T cells from mice of any strain and represents a valuable tool for preclinical immunotherapy studies. Here, we describe the superiority of γ-retroviral vectors compared with lentiviral vectors for transduction of murine T cells and surprisingly illustrate robust gene-transfer into phenotypically naive/memory-stem cell like (TN/TSCM; CD62L(hi)/CD44(low)) and central memory (TCM; CD62L(hi)/CD44(hi)) CD8+ T cells using murine stem cell-based γ-retroviral vectors (MSGV1). We created MSGV1 vectors for a major histocompatibility complex-class I-restricted TCR specific for the melanocyte-differentiation antigen, glycoprotein 100 (MSGV1-pmel-1), and a major histocompatibility complex-class II-restricted TCR specific for tyrosinase-related protein-1 (MSGV1-TRP-1), and found that robust gene expression required codon optimization of TCR sequences for the pmel-1 TCR. To test for functionality, we adoptively transferred TCR-engineered T cells into mice bearing B16 melanomas and observed delayed growth of established tumors with pmel-1 TCR engineered CD8+ T cells and significant tumor regression with TRP-1 TCR transduced CD4 T cells. We simultaneously created lentiviral vectors encoding the pmel-1 TCR, but found that these vectors mediated low TCR expression in murine T cells, but robust gene expression in other murine and human cell lines. These results indicate that preclinical murine models of adoptive immunotherapies are more practical using γ-retroviral rather than lentiviral vectors.

This study compared the efficacy of SPLAT-OrB, a new pheromone formulation for oriental beetle mating disruption that can be mechanically applied, with hand-applied plastic dispensers in commercial blueberry fields. Both formulations were tested at 2.5 and 5 g of the major sex pheromone component (Z)-7-tetradecen-2-one per hectare, and evaluated by measuring trap shut-down, mating success of caged females, and the number of grubs in sentinel blueberry pots baited with tethered females. All pheromone-treated plots had fewer male captures in traps and lower mating success of caged females compared with untreated plots. SPLAT-OrB, and plastic dispensers at the higher rate, also reduced the number of grubs in sentinel pots. To understand the mechanism of mating disruption in oriental beetle, males were observed approaching the pheromone sources in disrupted plots. In addition, male oriental beetle captures were quantified in plots treated with varying SPLAT-OrB dollop densities per ha. Consistent with predictions for competitive attraction, field observations revealed males approaching the pheromone source and male captures decreasing concavely with increasing dollop density. In a mark-release-recapture study, male oriental beetles responded to SPLAT-OrB dollops and plastic dispensers at least 60 m from the source. Additionally, SPLAT-OrB emitted pheromone that was attractive to male oriental beetles for >5 wk; however, emission rates and attraction dropped rapidly during the first 3-4 wk. This study demonstrates the feasibility of using SPLAT-OrB for oriental beetle mating disruption as an alternative to hand-applied plastic dispensers, and conclusively reveals that a principal mechanism is the competitive attraction of males., (© 2010 Entomological Society of America)

Immunotherapies based on adoptive cell transfer are highly effective in the treatment of metastatic melanoma, but the use of this approach in other cancer histologies has been hampered by the identification of appropriate target molecules. Immunologic approaches targeting tumor vasculature provide a means for the therapy of multiple solid tumor types. We developed a method to target tumor vasculature, using genetically redirected syngeneic or autologous T cells. Mouse and human T cells were engineered to express a chimeric antigen receptor (CAR) targeted against VEGFR-2, which is overexpressed in tumor vasculature and is responsible for VEGF-mediated tumor progression and metastasis. Mouse and human T cells expressing the relevant VEGFR-2 CARs mediated specific immune responses against VEGFR-2 protein as well as VEGFR-2-expressing cells in vitro. A single dose of VEGFR-2 CAR-engineered mouse T cells plus exogenous IL-2 significantly inhibited the growth of 5 different types of established, vascularized syngeneic tumors in 2 different strains of mice and prolonged the survival of mice. T cells transduced with VEGFR-2 CAR showed durable and increased tumor infiltration, correlating with their antitumor effect. This approach provides a potential method for the gene therapy of a variety of human cancers.

Adoptive cell transfer using autologous tumor infiltrating lymphocytes or lymphocytes transduced with antitumor T-cell receptor (TCR) is an effective therapy for patients with metastatic melanoma. A limiting factor in the effectiveness of this treatment is the apoptosis of the transferred cells when Interleukin-2 (IL-2) administration is withdrawn. In an attempt to improve persistence of the transferred lymphocytes, we cotransduced human peripheral blood lymphocytes with retroviruses encoding Bcl-2 or Bcl-xL, antiapoptotic genes of the BCL2 family, and the MART-1 melanoma tumor antigen-specific TCR, DMF5. Lymphocytes were cotransduced with 38% to 64% cotransduction efficiency, and exhibited a marked delay in apoptosis after IL-2 withdrawal. Cotransduction with Bcl-2 or Bcl-xL did not affect cytokine secretion or lytic ability of the DMF5-transduced lymphocytes. After 5 days of IL-2 withdrawal, cotransduced lymphocytes produced similar levels of IFN-γ per cell as DMF5-alone transduced lymphocytes in response to tumor cells. Cotransduction did not alter the phenotype of lymphocytes with respect to a panel of T-cell differentiation markers. In a mouse model of melanoma, adoptively transferred T cells transduced with Bcl-2 persisted better in vivo at the site of tumor, 13 and 21 days after adoptive transfer (P=0.0064 and 0.041, respectively), with evidence of enrichment of the Bcl-2-transduced population over time (P<0.0001). Thus, by coexpressing Bcl-2 or Bcl-xL with a tumor-specific TCR, we have engineered a lymphocyte that resists apoptosis owing to IL-2 withdrawal without altering its tumor-specific function or phenotype, and thus may show improved antitumor effectiveness in vivo after cell transfer.

Adoptive cell transfer (ACT)-based immunotherapies can mediate objective cancer regression in animal models and in up to 70% of patients with metastatic melanoma; however, it remains unclear whether the tumor vasculature impedes the egress of tumor-specific T cells, thus hindering this immunotherapy. Disruption of the proangiogenic interaction of vascular endothelial growth factor (VEGF) with its receptor (VEGFR-2) has been reported to "normalize" tumor vasculature, enhancing the efficacy of chemotherapeutic agents by increasing their delivery to the tumor intersitium. We thus sought to determine whether disrupting VEGF/VEGFR-2 signaling could enhance the effectiveness of ACT in a murine cancer model. The administration of an antibody against mouse VEGF synergized with ACT to enhance inhibition of established, vascularized, B16 melanoma (P = 0.009) and improve survival (P = 0.003). Additive effects of an antibody against VEGFR-2 in conjunction with ACT were seen in this model (P = 0.013). Anti-VEGF, but not anti-VEGFR-2, antibody significantly increased infiltration of transferred cells into the tumor. Thus, normalization of tumor vasculature through disruption of the VEGF/VEGFR-2 axis can increase extravasation of adoptively transferred T cells into the tumor and improve ACT-based immunotherapy. These studies provide a rationale for the exploration of combining antiangiogenic agents with ACT for the treatment of patients with cancer.

Tyrosinase, an enzyme involved in melanin synthesis, is expressed in nearly all primary and metastatic melanoma lesions and thus is an attractive target for TCR-based gene therapy using adoptive cell transfer. The TCR alpha- and beta-chain genes from a tumor-infiltrating lymphocyte, which recognized the tyrosinase 368-376 peptide in the context of HLA-A2, were cloned into a gamma-retroviral vector. Following transduction of PBL, specific reactivity was confirmed by cytokine production following coculture with tumor targets. Experiments using Ab blockade and CD4/CD8 sorting of the transduced PBLs demonstrated that this antityrosinase TCR was CD4/CD8 independent. The introduction of a second disulfide bond between the TCR constant regions and/or creation of a chimeric protein in which the human constant regions were replaced by murine homologs resulted in enhanced TCR expression as demonstrated by tetramer staining and improved tumor reactivity that was comparable to PBL transduced with either anti-melanoma Ag recognized by T cells-1 or anti-gp100 TCR vectors currently used in clinical trials. The chimeric TCR also allowed us to test antitumor function of in HLA-A2/K(b)-transgenic mice. Transfer of the antityrosinase TCR into mouse splenocytes conferred CD4/CD8-independent, HLA-A2-restricted Ag reactivity against B16/A2K(b) murine melanoma in vitro. Furthermore, adoptive transfer of transduced splenocytes mediated B16/A2K(b) melanoma tumor regression in lymphodepleted mice, and, surprisingly, both CD8 and CD4 T cells were equally effective in mediating tumor regression. These results suggest that this highly active tyrosinase-specific TCR could be of value in adoptive cell transfer for melanoma.

In the last decade lentiviral gene transfer vectors have gained significant place both in basic science and gene therapy applications. A number of gene transfer applications would benefit from vectors capable of expressing multiple genes. This chapter focuses on production of bicistronic and tricistronic lentiviral vectors based on the internal ribosomal entry site (IRES) sequence of encephalomyocarditis virus (EMCV) and/or foot-and-mouth disease virus (FMDV) cleavage factor 2A. Multigene vectors produced high titer viral particles and were able to simultaneously express two or three transgenes in transduced cells. The level of expression of individual transgenes varied depending on the transgene itself, its position within the construct, the total number of transgenes expressed, the strategy used for multigene expression, and the number of copies of proviral insertions.

Prostatic acid phosphatase (PAP) is a prostate cancer tumor antigen and a prostate-specific protein shared by rats and humans. Previous studies indicated that Copenhagen rats immunized with a recombinant vaccinia virus expressing human PAP (hPAP) developed PAP-specific cytotoxic T cells (CTL) with cross reactivity to rat PAP (rPAP) and evidence of prostate inflammation. Viral delivery of vaccine antigens is an active area of clinical investigation. However, a potential difficulty with viral-based immunizations is that immune responses elicited to the viral vector might limit the possibility of multiple immunizations. In this paper, we investigate the ability of another genetic immunization method, a DNA vaccine encoding PAP, to elicit antigen-specific CD8+ T cell immune responses. Specifically, Lewis rats were immunized with either a plasmid DNA-based (pTVG-HP) or vaccinia-based (VV-HP) vaccine each encoding hPAP. We determined that rats immunized with a DNA vaccine encoding hPAP developed a Th1-biased immune response as indicated by proliferating PAP-specific CD4+ and CD8+ cells and IFNgamma production. Rats immunized with vaccinia virus encoding PAP did not develop a PAP-specific response unless boosted with a heterologous vaccination scheme. Most importantly, multiple immunizations with a DNA vaccine encoding the rat PAP homologue (pTVG-RP) could overcome peripheral self-tolerance against rPAP and generate a Th1-biased antigen-specific CD4+ and CD8+ T cell response. Overall, DNA vaccines provide a safe and effective method of generating prostate antigen-specific T cell responses. These findings support the investigation of PAP-specific DNA vaccines in human clinical trials.

Fusion proteins consisting of the ligand-binding domain of CTLA4 covalently attached to an antigen (Ag) are potent immunogens. This fusion strategy effectively induces Ag-specific immunity both when introduced as a DNA-based vaccine and as a recombinant protein. CTLA4 is a ligand for B7 molecules expressed on the surface of antigen-presenting cells (APCs), and this interaction is critical for the fusion protein to stimulate Ag-specific immunity. We show that interaction of the fusion protein with either B7-1 or B7-2 is sufficient to stimulate immune activity, and that T cells are essential for the development of IgG responses. In addition, we demonstrate that human dendritic cells (DCs) pulsed with CTLA4-Ag fusion proteins can efficiently present Ag to T cells and induce an Ag-specific immune response in vitro. These studies provide further mechanistic understanding of the process by which CTLA4-Ag fusion proteins stimulate the immune system, and represent an efficient means of generating Ag-specific T cells for immunotherapy.

Objective: To describe the case of a person with schizophrenia and agenesis of the corpus callosum., Conclusion: A 24-year-old Caucasian woman with schizophrenia was incidentally found to have complete agenesis of the corpus callosum. A comprehensive neuropsychiatric assessment allowed management to be specifically tailored to the patient's unique clinical profile.

Lentiviral vectors are capable of efficiently transducing nondividing and slowly dividing cells, including hematopoietic stem cells, resulting in stable integration and sustained transgene expression. We constructed human immunodeficiency virus type 1-based self-inactivating lentiviral vectors to express either wild-type or an O6-benzylguanine (O6-beG)-resistant mutant form of the human O6-alkylguanine-DNA methyltransferase (MGMT; DNA-O6-methylguanine:[protein]-L-cysteine S-methyltransferase, EC 2.1.1.63) and transduced K562 and granulocyte colony-stimulating factor-mobilized human peripheral blood CD34+ cells. After transduction, K562 cells expressed high levels of MGMT as determined by Western blot, immunocytochemistry, and biochemical assay. A colony-forming survival assay showed significant protection against O6-beG plus 1,3-bis(2-chloroethyl)-nitrosourea (BCNU) or temozolomide (TMZ) toxicity. Similarly, a single transduction of CD34+ cells resulted in a 13- to 14-fold increase in the level of MGMT expression. In comparison with non-transduced cells, mutant MGMTP140K-transduced CD34+ cells showed significant resistance against the combined toxicity of O6-beG with either TMZ or BCNU: there was an approximately 9-fold increase in the survival of colony-forming cells as indicated by the IC50 values after O6-beG plus TMZ treatment and an approximately 5-fold increase in the case of O6-beG plus BCNU treatment. These results show that lentivirus-mediated expression of MGMTP140K can efficiently protect the hematopoietic compartment against the combined toxicity of O6-beG plus TMZ or BCNU., (Copryright Mary Ann Liebert, Inc.)

Dendritic cells (DCs) are potent antigen-presenting cells and are capable of activating naive T cells. Gene transfer of tumor antigen and cytokine genes into DCs could be an important strategy for immunotherapeutic applications. Dendritic cells derived from peripheral blood monocytes do not divide and are therefore poor candidates for gene transfer by Moloney murine leukemia virus (Mo-MuLV)-based retroviral vectors. Lentiviral vectors are emerging as a powerful tool for gene delivery into dividing and nondividing cells. A three-plasmid expression system pseudotyped with the envelope from vesicular stomatitis virus (VSV-G) was used to generate lentiviral vector particles expressing enhanced green fluorescent protein (EGFP). Peripheral blood monocyte-derived DCs were cultured in the presence of GM-CSF and IL-4 and transduced with lentiviral or Mo-MuLV-based vectors expressing EGFP. FACS analysis of lentiviral vector-transduced DCs derived either from normal healthy volunteers or from melanoma patients demonstrated transduction efficiency ranging from 70 to 90% compared with 2-8% using Mo-MuLV-based vectors pseudotyped with VSV-G. Comparison of lentiviral vectors expressing EGFP driven by CMV or human PGK promoters showed similar levels of transgene expression. Lentiviral vector preparations produced in the absence of HIV accessory proteins transduced DCs at efficiencies equal to vectors produced with accessory proteins. Alu-HIV-1 LTR PCR demonstrated the genomic integration of the lentiviral vector in the transduced DCs. Transduced cells showed characteristic dendritic cell phenotype and strong allostimulatory capacity and maintained the ability to respond to activation signals such as CD40 ligand and lipopolysaccharide. These results provide evidence that lentiviral vectors are efficient tools for gene transfer and expression in monocyte-derived DCs that could be useful for immunotherapeutic applications.

Resting lymphocytes are refractory to gene transfer using Moloney murine leukemia virus (MMLV)-based retroviral vectors because of their quiescent status. Recently, it has been shown that lentiviral vectors are capable of transferring genes into nondividing and terminally differentiated cells. We used human immunodeficiency virus type-1 (HIV-1)-based vectors expressing enhanced green fluorescent protein (EGFP) driven by different promoters (CMV, MPSV, or PGK) and investigated their ability to transduce human T- and B-cell lines, as well as resting or activated primary peripheral and umbilical cord blood lymphocytes. The effects of the presence or the absence of HIV-1 accessory proteins (Vif, Vpr, Vpu, and Nef) in the vector system were also assessed. Flow cytometry analysis showed no differences in the ability of these vectors of transferring the reporter gene into lymphocytic lines and mitogen-stimulated primary lymphocytes in the presence or the absence of HIV-1 accessory proteins (APs). Similarly, viral supernatants generated in the presence of accessory genes could efficiently transduce various subsets of resting lymphocytes and provide long-term expression of the transgene. No significant transduction-induced changes in cell activation or cycling status were observed and Alu-HIV-1 long terminal repeat polymerase chain reaction (LTR PCR) analysis demonstrated integration of the vector sequences at the molecular level. In contrast, in the absence of HIV-1 APs, lentiviral vectors failed to integrate and express the transgene in resting lymphocytes. These results show that transduction of primary resting lymphocytes with HIV-1-based vectors requires the presence of viral accessory proteins. (Blood. 2000;96:1309-1316)

Retroviral gene transfer was used to achieve expression in mouse bone marrow of a mutant form of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (hATPA/GA), which exhibits resistance to inactivation by O6-benzylguanine (O6-beG). After reconstitution of mice with transduced bone marrow, approximately 50% of the bipotent granulocyte-macrophage colony-forming cell (GM-CFC) and multipotent spleen colony-forming unit (CFU-S) hemopoietic populations showed expression of the transgene; this expression was associated with resistance to either mitozolomide or to a combination of O6-beG and mitozolomide, relative to mock-transduced controls. Thus, at a dose of mitozolomide in vivo that allowed only 70% and 62% survival of mock-transduced GM-CFC and CFU-S, respectively, the hATPA/GA CFC were totally resistant to the same dose of mitozolomide (P < .05 and .001, respectively). In the presence of O6-beG, the toxicity of mitozolomide was greatly potentiated. Only 24% and 18%, respectively, of mock-transduced GM-CFC and CFU-S survived combination treatment, whereas 45% (P < .05) and 37% (P < .01) of GM-CFC and CFU-S, respectively, from hATPA/GA mice survived the same combination of doses. Furthermore, as a result of transgene expression, the number of micronucleated polychromatic erythrocytes induced by mitozolomide was significantly reduced (P < .05) by 40% relative to mock-transduced controls, indicating the potential of this approach to reduce the frequency of mutation associated with chemotherapy exposure. The protection against the toxic and clastogenic effects of mitozolomide in both primitive and more mature hemopoietic cells suggests that the severe myelosuppression that halted further clinical investigation of this drug could be substantially ameliorated by the exogenous expression of O6-alkylguanine-DNA alkyltransferase. Therefore, these data raise the prospect for the reinvestigation of mitozolomide and other proscribed drugs in the context of genetically protected hemopoiesis.